A study on Clinical, Biochemical, Drug trough level and histopathological correlation of calcineurin inhibitor (CNI) toxicity in renal allograft recipient.

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“ A STUDY ON CLINICAL, BIOCHEMICAL, DRUG TROUGH LEVEL AND

HISTOPATHOLOGICAL CORRELATION OF CALCINEURIN INHIBITOR (CNI) TOXICITY IN

RENAL ALLOGRAFT RECIPIENT”

Dissertation Submitted to

THE TAMIL NADU DR.M.G.R.MEDICAL UNIVERSITY, CHENNAI- 600 032.

In partial fulfillment of the regulation for the award of the degree of

DM (NEPHROLOGY) BRANCH - III

MADRAS MEDICAL COLLEGE

RAJIV GANDHI GOVERNMENT GENERAL HOSPITAL CHENNAI – 600 003.

AUGUST 2014

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DECLARATION

I, Dr.Gandhimohan.R, solemnly declare that the dissertation TITLED “A STUDY ON CLINICAL, BIOCHEMICAL, DRUG TROUGH LEVEL AND HISTOPATHOLOGICAL CORRELATION OF CALCINEURIN INHIBITOR (CNI) TOXICITY IN RENAL ALLOGRAFT RECEPIENT” is the bonafide work done by me at Department of Nephrology, Madras Medical College under the expert guidance and supervision of Dr. N.GOPALAKRISHNAN M.D., D.M., FRCP, Professor of Nephrology, Madras Medical College. The dissertation is submitted to the Tamilnadu Dr.M.G.R Medical University towards partial fulfillment of requirement for the award of D.M. Degree (Branch III) in Nephrology.

Place: Chennai Dr. Gandhimohan.R

Date:

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CERTIFICATE

This is to certify that the dissertation entitled “A STUDY ON CLINICAL, BIOCHEMICAL, DRUG TROUGH LEVEL AND HISTOPATHOLOGICAL CORRELATION OF CALCINEURIN INHIBITOR (CNI) TOXICITY IN RENAL ALLOGRAFT RECIPIENT”is a bonafide work done Dr.Gandhimohan.R, Department of Nephrology, Madras Medical College, in partial fulfillment of the University rules and regulations for award of D.M., Nephrology under my guidance and supervision during the academic year 2011 – 14.

Dr. N.GOPALAKRISHNAN, M.D., D.M., FRCP, Prof.VIMALA.R.M.D.,

Professor of Nephrology, Dean,

Department of Nephrology, Madras Medical College, Madras Medical College, Chennai. - 3

Chennai.- 3.

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ACKNOWLEDGEMENT

I thank the Dean, Prof. Dr. Vimala. R M.D, Madras Medical College for permitting me to performing this study at the Nephrology department.

I express my sincere gratitude to Prof. Dr. V. Kanagasabai, M.D, former Dean, Madras Medical College, Chennai for allowing me to conduct this study at madras medical college.

I wish to express my sincere thanks to my most respected Chief Prof. Dr..N.Gopalakrishnan, M.D, D.M, FRCP, Professor and Head, Department of Nephrology, Madras medical college, Chennai for the constant guidance and support he rendered me throughout the study.

I am thankful to Dr. T. Balasubramanian, M.D, D.M, Associate Professor, Department of Nephrology for his valuable suggestions and guidance in doing this study.

I am immensely grateful to Dr.Malathy, Dr.Harris, Dr.N.D.Srinivasa Prasad, Dr.Shakthirajan, Dr.Dinesh, Dr.Danapriya for their valuable suggestions which helped me to model this study..

I thank all my patients without whose participation this study would not have been a reality.

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I thank all my colleagues, friends, technicians, and staff of the Department of Nephrology, Madras Medical College, Chennai, for their help and support they extended for the successful completion of this dissertation.

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Introduction

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INTRODUCTION

The purpose of renal replacement therapy for End Stage Renal Disease patients was to prolong and maintain quality of life. Despite the many attempts to do renal replacement in early part of 20^th century, the first successful renal transplant was done in 1954 by Murray among identical twins.

Introduction of calcineurin inhibitor in later part of twentieth century revolutionized the history of renal transplantation by reducing the short term morbidity and mortality. However the patients receiving calcineurin inhibitor were under the risk of calcineurin inhibitor nephrotoxicity in long run. The chronic nephrotoxic effects of calcineurin inhibitors associated with the renal parenchymal damage plays a major role in the pathogenesis of chronic renal dysfunction. Calcineurin inhibitor toxicity clinically characterized by tremor, hypertension, hypertrichosis and gum hypertrophy, biochemically by raising creatinine (graft dysfunction), hyperglycemia, hyperkalemia and hyperuricemia and histopathologicaly by isometric vacuolization, arterial nodular hyalinosis, striped fibrosis and interstitial atrophy.

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The effect of toxicity was reversible in short term, became irreversible in long term. Lower dose results in graft dysfunction and rejection, higher dose results in toxicity because of its narrow therapeutic index ( little difference between toxic and therapeutic doses).

So it was mandatory to adjust its dosage according to measurements of the actual blood levels through therapeutic drug monitoring (TDM). The serum level of drug does not correlat with the degree of nephrotoxicity in most of the occasion because of its varied pharmacokinetics, narrow therapeutic index, individual sensitivity to toxic effects. .

Though there are few international studies on prevalence of calcineurin inhibitor toxicity and its clinico pathological correlation, a good study in this part of world is lacking . So, this study, attempt to find out the correlation among clinical, biochemical, drug trough level and histopathological features of calcineurin inhibitor toxicity.

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Aim of the

Study

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AIM OF THE STUDY

To study the clinical, biochemical, whole blood trough level and histopathological correlation of calcineurin inhibitors (CNI) toxicity in renal allograft recipients

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Review of

Literature

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CALCINEURIN INHIBITORS

Perhaps the most effective immunosuppressant drug, which served as the back bone of kidney transplant for past 2-3 decades. These comprise of Cyclosporine and Tacrolimus, although they are structurally distinct and combined with distinct immunophilin, cyclophilin and FK Binding Protein which act through common path way by inhibiting dephosphatase enzyme Calcineurin.

Calcineurin catalyzed dephosphorylation was required for entry of cytoplasmic component of nuclear factor of activated T cells into nucleus where it combined with its nuclear counter part and induces number of cytokine genes, especially interleukin–2, which play a major role in activation, differentiation and proliferation a T cells.

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CALCINEURIN INHIBITORS-MECHANISM OF ACTION

In cytoplasm, cyclosporine binds to a cis-trans-peptidyl-prolyl- isomerase. Which was important in, folding proteins and peptides in to their native conformation (Immunophilin-protein that binds immunosuppressive agent: cyclophillin binds cyclosporine; FK-binding protein binds tacrolimus and rapamycin)¹. Calcineurin-Immunophilin complex (i.e, cyclosporine- cyclophilin, tacrolimus – FK binding protein) binds to a calcium and calmodulin dependent phosphatase calcineurin.

Which plays crucial role in transduction of calcium dependant signal.

Calcineurin, a phosphatase enzyme, which normally dephosphorylate the cytosolic part of nuclear factor of activated T cells in order to its entry into nucleus² and combined with nuclear part of activated T cells, which activates the promoter region of Interlukin L- 2(IL-2) leading to its transcription³, which results in reduction in its production, expression on cell surface and the resultant reduction in T cell activation and proliferation. Apart from its reduction in Interlukin-2 production, it also impairs the transcription of Interlukin-4(IL-4), Interferon(IFN)-gamma and tumour necrosis factor(TNF)-alpha.

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The transcription of other genes, such as CD-40 ligand and the proto oncogenes H-ras and C-myc is also impaired. The stimulation of proto oncogene may be relevant to the cause of certain post transplant neoplasia.

Cyclosporine enhances the mRNA expression of Transforming Growth Factor (TGF)-beta in activated T cells⁴ and constrain new DNA Synthesis. Patients on cyclosporine were found to have higher level of TGF-beta than patients on other immune suppressive drugs. TGf-beta inhibits IL-2 dependent, T Cell activation, and suppress the antigen specific T Cell proliferation. TGF-beta type 1 was a prosclerotic which play a major role in chronic cyclosporine nephrotoxicity. Cyclosporine apart from increasing TGF-beta, it increases the expression of its receptor in mesangial cells and activates the production of plasminogen activator inhibitor and fibronectin. Islem et al showed that anti TGF-beta antibodies prevents the certain changes of cyclosporine induce chronic nephrotoxicity. Thus the TGF-beta had the immunosuppressive role on its own and mediates immune suppressive effects of cyclosporine.

Thus the TGF-beta may play a central role in mediation of beneficial and detrimental effects of calcineurin inhibitor. Dentritic cell plays a major role in antigen presentation. Cyclosporine inhibits its migration, maturation, and impairs its antigen presenting capabilities⁵.

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CYCLOSPORINE

Cyclosporine is a small cyclic polypeptide of 11 amino acids in position 1, 2, 3, & 11 and molecular weight of 1200Kd. The cyclic polypeptide structure was necessary for its action. Cyclosporine is soluble only in lipids and organic solvents. First isolated by the department of microbiology at Sandoz (Switzerland) from two strains of imperfect fungai Cylindeocarpon lucidum booth and Trichoderma polysporum rifai as an antifungal agent⁶.

Formulations

Cyclosporine available in oral and intravenous preparations. The original oil based sand-immune preparation had been replaced by microemulsion(Neo-oral). The oral preparation available in solution and soft gelatin capsules. Oral sand immune prepararion has variable time to peak(Cmax) concentration but averages 4 hours. Second peak appears in substantial portion of transplant patients. The oral bioavailability of cyclosporine was better. The peak cyclosporine level(Cmax) was higher and trough(Co) concentration correlates better with the systemic exposure as reflected by the area under the curve(AUC). Generic formulations of cyclosporine were available. FDA approved the generic formulations. But because of its varied absorption and narrow therapeutic index the bioequivalence stantardization of FDA for cyclosporine was more

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rigorous. While changeover from branded to generic formulations, the drug level monitoring should be intensified. Since the pharmacokinetics and bioequivalence were different for sand immune and neo-oral its generic formulation should not be interchanged. Cyclosporine absorption was delayed and decreased by food. High and low fat meals decreases the area under the curve by 13% and maximum concentration by 33% when it consumed with in thirty minutes of drug intake. Cyclosporine distributed extensively in extra vascular compartment, at steady state the volume of distribution was 3-5L/Kg in kidney transplant recipients after Intra venous dosing.

Factors determining absorption

Individual patient Type of transplant Transplant age Bile flow

Gastro Intestinal motility state Type of formulations

Distribution

In blood, one third was bound to lipoproteins in plasma, remaining two third found in RBCs. So the whole blood levels were higher than plasma. Cyclosporine which bound with lipoproteins were easily enters

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plasma membrane. Hence hypo cholesterelemia exaggerates toxicity and heper cholesterelemia reduces its toxicity. Hyperlipidemia was due to its affinity towards LDL receptor.

Metabolism

Cyclosporine was extensively metabolized by cytochrome P 450 3A in liver and gastrointestinal microsomal enzyme system⁶. It underwent first pass metabolism in liver and gastrointestinal tract by cytochrome P 450 3A and P-glycoprotein. The variability in its metabolism among individual was due to polymorphism⁷in cytochrome P 450 3A and P-glycoprotein. More than twenty metabolites of cyclosporine had been identified in bile, blood, feces and urine.

Metabolites were inactive when compared to parent drug. Six percent of metabolites were excreted in urine. Only 0.1% of cyclosporine was excreted unchanged in urine. Since cyclosporine was neither excreted in urine nor removed by dialysis dose modification does not required in renal dysfunction. Cyclosporine was secreted in breast milk but in smaller quantity. Majority of its metabolites excreted through bile by liver, hence dose modification was imperative in liver disease. Half life was biphasic, averages 8.4-27 hours(range 4-50).

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TACROLIMUS

Tacrolimus was a macrolide immuno suppressant isolated from fungus Streptomyces tsukubaensis in 1994 from Japan⁸. It was a 23 membered macrolide lactone. It was a neutral and hydrophilic compound.

Initially it was used in liver transplant only. Approved by a FDA for kidney transplant in 1997, within a decade of approval its use had been raised to 67% in kidney transplant because of its potential immuno suppressive action.

Absorption

Tacrolimus absorption was variable. Bio availability varies from 5% to 95% (mean 25%). Reasons for reduced absorption were african/non-caucasians, diabetic patients and fatty food. Peak concentration attained after 0.5-1 hour. While took with food peak concentration delayed by 50-25% and the area under curve decreased by 25-40%. In intestine absorbed tacrolimus was metabolized by CYP P 450 3A, extruded into intestinal lumen by P-glycoprotein. Extruded drug again get reabsorbed⁹. Bile is not essential for tacrolimus absorption.

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Distribution

In blood, tacrolimus was intensively bound to erythrocytes. Whole blood drug concentration significantly higher (4-14 time) than corresponding plasma level. In plasma most of the drug bound with alpha1 acid glycoprotein, globulin and albumin. In pediatric recipients a volume of distribution was higher than adult, because of increased permeability of membrane and reduced quality and affinity to plasma protein.

Metabolism

Tacrolimus was extensively metabolized by cytochrome P 450 3A4 in liver and intestinal epithelial cells by hydroxylation and demethylation. Metabolism of tacrolimus was highly variable because of cytochromeP 450 3A4 polymorphism. Expression of cytochrome P 450 3A4 varied from 10-100 fold in liver and 30-40 fold in intestine. The metabolites were one-tenth as active as tacrolimus. Its metabolites were seen in urine, feces and bile.

Elimination

More than 95% of tacrolimus was eliminated in bile¹⁰. Cholestasis increases the drug level. Urinary excretion accounts for 2.4%.

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Factors affecting the pharmacokinetics of tacrolimus Special patient population

In renal transplant, tacrolimus clearance was higher among live related kidney transplant recipient than cdaver transplant recipient because of low haematocrit and albumin. Diabetic patients shown 38%

reduction in area under curve because of altered gut motility. Cystic fibrosis patients with pancreatic involvement in need of 40% higher dose because of decreased absorption due to pancreatic enzyme deficiency.

Hepatic dysfunction

Tacrolimus clearance was reduced up to 2-3 fold in patients with liver dysfunction. In hepatitis C virus infection drug level was higher because of alrtered cytochrome P system.

Renal function

Tacrolimus clearance was not altered by renal dysfunction and dialysis.

Age

Children required higher dosage because of differences in cytochrome P 450 3A, bowel length and P glycoprotein expression.

Sex

No difference between sex.

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Race

African requires more dose than caucasians due to differences in expression of cytochrome P4503A and P glycoprotein expression in intestine and liver.

Haematocrit and albumin:

Reduction in haemotocrit and albumin results in lesser concentration of drug in whole blood.

Diurnal variation

Area under curve after morning dose was more than area under curve after night dose because of circadian effect on gastric emptying time and gastro intestinal perfusion.

Food

Effect of food depends on fat contents of food. Low fat content may delay the C-max.

Steroid

Steroid may induce cytochrome P 4503A and tacrolimus metabolism.

Diarrhea

Increased tacrolimus level because of loss of P glycoprotein which prevent extrusion of drug from gastro-intestinal epithelial cells.

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PHARMACOGENETICS OF CALCINEURIN INHIBITORS(CNI)

Variability in cyclosporine Pharmacokinetics among individual was due to variability in exposure and function of cytochrome P 450 3A4 and P glycoprotein polymorphism¹¹. P-glycoprotein polymorphism was due to ABCB1 gene Polymorphism. P-glycoprotein, a membrane protein found on membrane, act as exporter of intracellular xenobiotics, which is ATP dependent. In kidney P glycoprotein expressed on the luminal surface of proximal and distal tubular epithelial cells.

Cyclosporine act as substrate for P glycoprotein, so if there is any defect in expression or function of P glycoprotein due to ABCB1 gene polymorphism cyclosporine accumulate inside the cell and lead on to nephrotoxicity¹². According to Anglichem et al, sirolimus and cyclosporine competes for P glycoprotein. So if used in combination sirolimus cause cyclosporin accumulation and nephrotoxicity.

Transcriptional analysis revealed that epithelial mesenchymal trans formation(EMT) and endoplasmic reticulum stress are the two main mechanism which cause CNI nephrotoxicity. In vitro studies revealed that cyclosporine induced EMT changes in proximal tubular epithelial cells through TGF-beta up regulation. Vimentin expression had been increased in rats treated with cyclosporine.

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Endoplasmic reticulum stress is due to accumulation of mis-folded proteins within endoplasmic reticulum. It has been proved by in vitro studies. Progress in whole genome studies, molecular biology and functional genetics will throw further light on this area in future.

CALCINEURIN INHIBITORS-DRUG INTERACTION

Cyclosporine was degraded by hepatic cytochrome P450. Durgs which enchances the ability of cytochrome P450 reduces the concentration of cyclosporine and drugs that inhibit the cytochrome P450 increases the concentration of cyclosporine level¹³ in blood.

Durgs that decreases the calcineurine level (cytochrome P450 inducer)

:

Anti tuberculous drugs

Rifampicin ( marked reduction ) Pyrizinamide

Ethambutol Anticonvulsants

Barbiturates (marked reduction )

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Phenytoin Primidone

Carbamazepine (mild reduction) Modafinil (mild reduction)

Oxcarbazepine (second generation – mild reduction) Antibiotics:

Nafcillin

Intravenos trimethoprim Intravenos sulphadimidine.

Imipenem Cephalosporin Terbinafine Others:

St.John’s wort (Hypericum perforatum) – Herbal anti depressant

Ticlodipine.

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Corticosterods

Drugs that increases calcineurin level(cytochrome P450 inhibitor):

Calcium channel blocker:

Verapamil (40% reduction in CNI dose) Diltiazem (40% reduction in CNI dose) Amlodipine

Nicardipine

Nifedepine (minimal effect) Felodipine (minimal effect) Anti fungal

Ketoconazole (80% reduction of CNI dose) Fluconazone

Itraconazole Voricanozole Antibiotics:

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Erythromycin Clarithromycin

Azithromycin (conflicting reports) Anti retroviral drugs:

Protease inhibitor (Ritonavir) Hormones:

Oral contraceptive Anabolic steroids Testosterone Nor ethisterone Danazol

Somatostatin Other drugs:

Amiodarone Carvedilol Allopurinol

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Bromocriptine Chloroquine

Grape fruit juice (cytochrome P450 inhibitor) Drugs that increases absorption:

Metoclopromide Grape fruit juice

Drugs that exaggerates CNI nephrotoxicity.

Potentially nephrotoxic drugs should be avoided while patient on calcineurine inhibitor. Can be used with appropriate monitoring

Amphotericin Aminoglycoside NSAID

ACEI / ARB

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CALCINEURIN INHIBITORS TOXICITY

Despite the advantages of calcineurin inhibitors in solid organ transplantation and other diseases, its side effects hampers the long term graft and recipient morbidity and mortality¹⁴.

Renal

Acute calcinurin inhibitor nephrotoxicity Chronic calcinurin inhibitor nephrotoxicity Hepatic

Neurologic Cardiovascular Dermatologic Dental

Metabolic

Lipid abnormality

New Onset Diabetes Mellitus Hyperuricemia and Gout

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Infection and malignancy Thromboembolism

Renal toxicity

Acute calcineurin inhibitor nephrotoxicity Acute arteriolopathy

Tubular isometric vacuolization Thrombotic microangiopathy

Chronic calcineurin inhibitor nephrotoxicity

Interstial fibrosis and tubular atrophy (typically stripped) Medial arteriolar hyalinosis

Glomerular capsular fibrosis Global glomeruloselerosis

Focal segmental glomerulo sclerosis Juxta glomerular apparatus hyperplasia Tubular micro calcification

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Nephrotoxicity was the major side effect of these drugs. Initial experiences revealed that it was reversible hemodynamic changes. In 1984 Meyers et al demonstrated that progressive and irreversible changes also occurs in kidney resulting in tubular and interstial injury and glomerulosclerosis. The reversible changes were known as acute calcineurin inhibitor nephrotoxicity. Irreversible changes were known as chronic calcineurin inhibitor nephrotoxicity

Acute arteriolopathy (vascular effect

)

In 1985, Murray et al shown that afferent arteriolar constriction¹⁵ was caused by cyclosporine, which result in acute reversible functional impairment of glomerular filtration. This is due to imbalance between vasoconstrictor and vaso dilator substances produced by cyclosporine (vasoconstrictor : endothelin, rennin angiotensin ; vasodilator : prostacyclin, prostaglandin E2, nitrous oxide) and free radical formation.

Endothelin was a potent vaseconstrictor widely released in the kidney and vascular bed. The role endothelin in acute reversible vasoconstriction was established by the obliteration of these vasoconstrictive effect by anti endothelin antibodies¹⁶. Apart from direct afferent arteriolar vasoconstriction it also stimulates the rennin angiotensin system by recruiting rennin secreting cells in juxta glomerular apparutus and results in increased rennin production. Rennin enhances angiotensin II and

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reduces blood flow. Thus it becomes vicious cycle. On chronic use of cyclosporine it results in juxta glomerular cells hyperplasia. The molecular mechanism by which cyclosporine recruits the renin secreting cells in afferent arterioles and increases renin secretion was currently not known. Cyclosporine and tacrolimus inhibits endothelium mediated nitrous oxide synthesis thus by inhibits vasodilation. Cyclosporine induced vasooclusion results in hypoxia, free radical formation and super oxide production. By farming peroxynitrite, super oxide decreases nitrous oxide bio availability.

Tubular effects (toxic tubulopathy)

Histologically isometric vacuolization¹⁷in tubular epithelial cells by cyclosporine was due to enlargement of endoplasmic reticulum and increased lyzosomes. These vacuolization was found with cyclosporine and tacrolimus in the absence of renal dysfunction. Renal dysfunction may found in the absence of morphological feature. Recent studies revealed that these vacuolization also occurs in renal ischemia (on tubular epithelial injury due to intra venus administration of hyper osmotic fluid (manitol, inulin, glucose). These vacuoles were varying in size (in contrast to calcineurin induced) and called as osmotic nephrosis. Apart from ischaemic insult direct role of calcineurin inhibitors also postulated, inclusion bodies were also expressed as on tubular epithelial cells, which

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are nothing but giant mitochondria and auto lysozomes. Even though mechanism of inclusion body formation was not known the important effect of cyclosporine on mitochondrial function was proved. How ever inclusion bodies also found in ischemic injury and preimplantation donor biobsies.

Thrombotic microangiopathy

It was a distinct form of calcineurin inhibitor nephrotoxicity, uncommon but serious histopathologically characterized by intra capillary platelet thrombi, intimal wall thickening, necrosis and luminal occulusion. This lesions may in patchy distribution and variable severity.

Mechanism postulated was endothelial damage caused by ischemia due to direct calcineurin inhibitor effect on endothelium. The concomitant increased platelet aggregation, plasminogen activator inhibitor activity and pronecrotic activity of calcineurine inhibitor on endothelium eventually lead to development of thrombotic micro angiopathy.

Chronic CNI nephrotoxicity

Calcineurin inhibitors not only induces acute reversible nephrotoxicity but associated with chronic reversible nephrotoxicity, which involves vessels (arterial hyalinosis), interstitium (interstitial fibrosis & tubular atrophy) and glomeruli (focal segmental glomerulo

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selerosis, thickening & fibrosis of bowman’s capsule). Nankivell et al shown that 15 years after transplantation lesions suggestive of irreversible damage were seen in all recipient. However these features were may be due to rejection, infection, hypertension, diabetes mellitus, drugs and aging. Since this study did not have control arm. Cycloscorin related haemodynamic changes and direct toxicity was thought to play a role.

Vascular effect

Afferent arterioles nodular hyalinosis was regarded as a hall-mark of CNI nephrotoxicity. Which was due to replacement of reactive smooth muscle cells by focal or circular protein (hyaline) deposits at the peripheral part of afferent arteriole wall, eventually results in narrowing of afferent arteriolar lumen. The molecular mechanism behind this was not well elucidated.

Tubulo-interstitial effect

Cyclosporine induced luminal narrowing, hypoperfusion, hypoxia and formation of reactive oxygen species results in cell death by apoptosis. Catalase, a enzyme which catalyse reactive oxygen species, antagonize the effects of cycloscorine induced apoptosis in vitro. Second hypothesis was the direct injury to the epithelial cell by cycloscorine

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which results in accumulation of intra-cellular reactive oxygen species, lipid peroxidation products along with an altered glutathione redox state.

Cyclosporine induced upregulation of TGF – beta play a major role in the formation of chronic interstitial fibrosis and tubular atrophy. TGF – beta promotes interstitial fibrosis by increasing the production of extra cellular matrix protein and decreasing the degradation. In addition TGF - beta promotes epithelial mesenchymal transition, which results in loss of epithelial phenotype. Which results in loss of epithelial polarization, denovo expression of dysregulated acting over smooth muscle, loss of intercellular adhesion through down regulation of E-cathedrin, destruction of basement membrane and increased cell invasiveness.

Remuzzi et al shown that hyper aldosteronism secondary to salt depletion and RAS activation increases the production of TGF – beta and reactive oxygen species, which cause interstitial fibrosis. Apoptosis of tubular and interstitial cells aggravated by direct toxic effect of CNI on apoptosis gene. In addition cyclosporine competitively inhibit the P-glycoprotein on luminal side of tubular epithelial cells and cause accumulation of toxic substances inside the cell.

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Glomerular effects

Chronic CNI intake results in glomerulosclerosis secondary to ischaemia induced by arterial hypertension. Tubular damage results in atubular glomeruli which was shrunken in size and shows periglomerular fibrosis. In addition CNI can cause secondary focal segmental glomerulosclerosis due to either arteriolar hyalinosis or global glomerulosclerosis.

Non specificity of histlogic findings

Morphological findings in CNI toxicity were not specific to CNI. It also found in other diseases. So called specific lesions such as tubular vacuolization and arterial hyalinosis also seen in other diseases.

Hypertension

Impaired renal haemodynamics due to afferent arteriolar vasoconstriction by CNI results in sodium, water retention and hypertension. In addition stimulation of sympathetic system, activation of RAS and suppression of atrial natriuritic peptide impairs the diuretic and natriuretic response to volume overload. Hypertension was tend to be less marked in tacrolimus on comparison with cyclosporine primarily due to less peripheral vasoconstriction.

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Hyperkalemia

Hyper/normokalemia, mild hyperchloremic acidosis and patients intact ability to secrete acid urine was usual features of CNI toxicity. It was due to inability to excrete acute potassium load due to defect in production of aldosterone or to its post receptor response. Exaggerated by concomitant acetyl choline esterase inhibitors, angiotension receptor blocker.

Hypomagnesemia & hypocalcemia

It was due to increased urinary loss in patients on CNI due to down regulation of specific transport proteins.

Hyperuricemia

Impaired uric acid secretion due to cyclosporine induced direct tubular defect which may lead on to gout.

Hepatic

Mild transaminites and mild hyper bilirubenemia may occur in 50% of patients on cyclosporine, due to defect in bile secretion, there may be any morphological changes. Cyclosporine use was associated with cholelithiasis due to increased lithogenecity. Gastro intestinal side effects such as anorexia, nausea, vomiting, diarrhea and abdominal discomfort occurs more with tacrolimus than cyclosporine.

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Neurologic

Coarse tremor, dysesthesia, headache, insomnia (dose and blood level related)

Cognitive impairment coinciding with peak drug level.

Occasional seizure, full blown leukoencephalopathy and bone pain.

Disabling pain, hallucination, seizure, cerebellar ataxia and motor weakness.

Dermatological

Hypertrichosis – obvious in dark haired girls.

Eye brow prominence

Dental

Gingival hyperplasia due to fibroblast proliferation and collagen deposition was common in cyclosporine and exaggerated by poor oral hygiene and concomitant calcium channel blocker. Severe gingival hypertrophy may require gingivectomy and switch to tacrolimus

Metabolic

Glucose intolerance

Impaired glucose tolerance and new onset diabetes mellitus was more common with tacrolimus than cyclosporine, reason being the more concentration of FK binding protein than cyclophilin in islets,. Steroid

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may exaggerate the cyclosporine induced glucose intolerance. Glycosuria may be result in direct cytoplasmic injury to tubules. Risk factors were obesity, family history of diabetes and hepatitis C virus.

Hyperlipedemia

Cyclosporine was implicated in post transplant hypercholesterolemia. The mechanisms were LDL receptor binding by cyclosporine, defective LDL feed back control by liver and altered bile acid synthesis.

Hyperuricemia

Cyclosporine induced tubular injury results in impaired uric acid secretion which results in accumulation of uric acid in blood and gout. It was aggravated by concomitant diuretic use. This was more with cyclosporine than tacrolimus.

Infection and malignancy

Infections and malignancies were inevitable following immunosuppressant use in organ transplantation. Infection more coincides with level of drugs in blood. More incidence of skin cancer had been reported with cyclosporine use. Cyclosporine can promote tumor progression, which was independent of its effects on the immune response.

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MONITORING OF CALCINEURIN INHIBITORS

Monitoring of cyclosporine level was essential, due to inter and intra patient variability, lower level results in rejection, higher lever results in toxicity, co prescribed drugs may result in alteration of cyclosporine level. Recovery from uraemia in early post transplant period results in increasing absorption so it needs close monitoring. Because of availability of various assay methods, option of multiple matrices (plasma, whole blood) and variable correlation with time of drugs intake results in much confusion.

Trough monitoring

Even though monitoring at the trough, before administering next dose (Co level) was traditional and convenient, the efficacy was questionable. Area under curve calculation using multiple blood sample assessment will be more effectively reflect the exposure of patient to drugs. Correlation with nephrotoxicity was not linear. Correlation with episode of acute rejection was also poor¹⁸.

Area under curve(AUC)

Even though calculation of AUC by assessing multiple blood samples in 0-12 hrs was more accurate, the process was largely impractical. Gaspais et al shown that AUC by 1, 5, 8 & 11hrs sample allowed accurate monitoring. Malhati et al shown that since microemulsion form of cyclosporine absorption variability limited to first

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four, the AUC 0-4 hrs, will correlate with clinical outcomes. But, it also requires multiple sampling. So the ideal strategy should be single point measurement. Monitoring two hours level (C2) was considered an ideal surrogate marker²⁰.

Two hours monitoring

Most accurate one point prediction of AUC 0-4 hrs, was the C2

level (sample taken two hours after last dose) and it showed less variability than either Co or C1 according to international renal transplantation study group. Canadian neoral renal transplantation study group’s result shown that C2 level of more than 1500 micro gram per liter at 2 weeks of post transplant period correlate significantly with lower rejection rates. According to Helsinki groups the rate of acute rejection was not significantly influenced by either C0 or C2. C2 Monitoring allowed a dose reduction in 34% of patients compared to 14.3% of patients in Co monitoring. In spite of dose reduction there was no improvement in renal function during 40 months follow up. Despite those controversial issues at the moment, trough (C0) remain the standard method of monitoring.

Cyclosporine assays

:

High perfusion liquid chromatography(HPLC) method:

Despite the availability of several methods to measure the Cyclosporine level HPLC was the gold standard, because of its ability to

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measure parent compound only. However it was expensive, labour intensive and not available in all centers. Its accuracy in lower plasma level was less précised.

Immunoassay:

1. Non specific polyclonal immunoassay.

2. Non specific monoclonal immunoassay.

Immuno assay are based on monoclonal / polyclonal anti bodies against cyclosporine. Monoclonal immune assay were largely replaced HPLC because it can be done on automated chemistry analyzers.

Fluorescence Polarization Immune Assay was the most common immune assay used now a days. But it overestimate by 45% because significantly cross reacts with cyclosporine metabolites.

Rapid Liquid Chromotography – mass spectrometry method was a newer method, which had been used in oxford laboratory, which eliminate the low precision of immune assay in lower level of concentration.

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Materials and

Methods

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MATERIALS AND METHODS

Study design

: Prospective study.

Inclusion criteria:

All end stage renal disease patient who underwent renal transplantation between June 2012 and June 2013 in the department of nephrology, Rajiv Gandhi government general hospital were included.

Exclusion criteria:

Patients who had Graft dysfunction due to surgical issues, who underwent graft nephrectomy, who expired were excluded.

Eligible patients were enrolled. Their demographic profile such as age, sex, type of native kidney disease, type of donor whether live related or deceased, age and sex of donor, type of relation in case live related were collected. In immediate post operative period if the patient have raising creatinine, complete blood count, peripheral smear study, platelet count, serum lactic dehydrogenase, urine analysis, serum electrolytes, liver function test, urine and blood culture sensitivity were done. USG KUB, Renal Doppler and necessary investigations were done in order to rule out surgery related complication.

Whole blood cyclosporine/tacrolimus trough(CO) level was assayed twelve hour after previous dose If the renal dysfunction does not

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35

due to surgical complication and patient showed clinical feature of Calcineurin toxicity such as tremor, paresthesia, hypertension(worsening hypertension in need of more drugs and new onset hypertension), paresthesia, gum hypertrophy, hypertrichosis, sodium, potassium, cholesterol were done.

In this study cyclosporine trough level was assayed by enzyme linked microparticle immune assay(EMIA) and tacrolimus trough level was assayed by chemiluscent enzyme linked immune assay(CLEIA).

As per our department protocol for those receive induction treatment cyclosporine level was considered in therapeutic range if it was 200-250ng/ml at 0-1month, 100-200 ng/ml at 2-6 months, and around 100 ng/ml after 6months. For tacrolimus, the level was considered in therapeutic range if it was around 8 ng/ml at 0-1 month, 5-6 ng/ml at 3- 6months, 3-5 ng/ml after 6months. For those didn’t receive induction, cyclosporine level was considered in therapeutic range if it was 200- 250ng/ml at 0-1month, 150-200ng/ml at 2-6 months, and around 100-150 ng/ml after 6months. For tacrolimus, the level was considered in therapeutic range if it was around 8-10ng/ml at 0-1 month, 7-8ng/ml at 3- 6months, around 5ng/ml after 6months

(46)

Results and

Analysis

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36

RESULTS

This study was conducted in our department among End Stage Renal Disease patients who underwent Renal Transplantation from June 2012 to June 2013. A total of sixty one patients were included out of seventy five. Of which males were 83.6% (51) and females were 16.4%

(10), (Table 1).

Table 1 Recipient sex

Sex No %

Male 51 83.6

Female 10 16.4

83.6 16.4

Sex of Recipient

Male Female

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37

Among Recipients majority of them were in 3^rd decade 39.3% (24), followed by equal proportion in 4^th 24.6% (15) and 5^th decade 24.6%

(15), (Table 2). Mean age was 31.45(range: 17-56years).

Table 2

Recipient age group in years

Age group No %

10-19 3 4.9

20-29 24 39.3

30-39 15 24.6

40-49 15 24.6

50-59 4 6.6

0 5 10 15 20 25 30 35 40

10-19 20-29 30-39 40-49 50-59

4.9

39.3

24.6 24.6

6.6

Recipient age group in years

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38

70.5% patients were live related donor recipients and 29.5% were deceased donor recipients. (Table 3).

Table 3 Type of Donor

Type of donor No %

Live donor 43 70.5

Deceased donor 18 29.5

Among live related donor males were 16.7% (7) and females were 83.3% (36) (Table 4).

Table 4

Sex Ratio of Live Donor

Sex No %

Male 7 16.7

Female 36 83.3

Of which mothers were 44.2% (19), followed by spouse 30.2% (13), fathers 16.3% (7) and sisters 9.3%(4), (Table 5).

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39

Table 5 : Relationship among Live donor

Relation No %

Father 7 16.3

Mother 19 44.2

Spouse 13 30.2

Sister 4 9.3

Among deceased donor males were 19.4% (17) and females were 5.6% (1), (Table 6).

0 5 10 15 20 25 30 35 40 45

Father Mother Spouse Sister

16.3

44.2

30.2

9.3

Relationship among Live donor

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40

Table 6

Deceased donor sex

Sex No %

Male 17 19.4

Female 1 5.6

After renal transplantation recipients were treated with immunosuppressive agents [three drugs : calcineurine Inhibitors (cyclosporine / Tacrolinus) + mycophenolate Mofetile / Azathioprine + Steroids] as per our department protocol. Induction treatment was given as per our department protocol [rabbit Anti Thymocyte Globulin / Interleukin – 2 receptor Antagonist) for high risk recipients such as those received organ from deceased donor, spouse donor and second transplant). These patients were followed up for 0-18 months (mean 12.5

19.4 5.6

Deceased donor sex

Male Female

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41

months) for calcineurin inhibitor toxicity. Observed toxic features were grouped into 0-3, 3-6, 6-9 and more than 9 months of post transplant age.

Follow up revealed that toxicity of calcineurin inhibitors were clinically present in 60.7% (37) in 0-3, 39.3% (24) in 3-6, 25.4%(15) in 6-9 months and 23.3%(10) in more than 9 months(Table 7).

Table 7

Clinical CNI toxicity Clinical

toxicity

0-3 months 3-6 months 6-9months >9months

n % n % n % n %

Yes 37 60.7 24 39.3 15 25.4 10 23.3

No 24 39.3 37 60.7 44 74.6 33 76.7

Among them, further toxicity profile was evaluated and grouped into 0-3, 3-6, 6-9 and >9 months. Evaluated toxicity profile were tremor,

0 10 20 30 40 50 60 70 80

0-3 Months 3-6 Months 6-9 Months >9 Months 60.7

39.3

25.4 23.3

39.3

60.7

74.6 76.7

Yes No

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42

paresthesia, hypertension(new onset /worsening), hypertrichosis, gum hypertrophy and NODAT(based on ADA guidelines) (Table 8).

Table 8

CNI Toxicity Profile

0-3 months 3-6 months 6-9months >9months

n % n % n % n %

Tremor Yes 24 39.3 21 34.4 15 25.4 9 20.9

No 37 60.7 40 65.6 44 74.6 34 79.1

Paresthesia Yes 2 3.3 1 1.6 0 0 0 0

No 59 96.7 60 98.4 59 100 43 100

Hypertension Yes 24 39.3 19 31.1 8 13.6 3 7

No 37 60.7 42 68.9 51 86.4 40 93

Hypertrichosis Yes 0 0 0 0 1 1.7 3 6.9

No 61 100 61 100 60 98.3 40 93.1

Gumhypertrophy Yes 0 0 0 0 1 1.7 1 2.3

No 61 100 61 100 60 98.3 40 97.7

NODAT Yes 3 4.9 3 4.9 4 6.8 1 2.3

No 58 95.1 58 95.1 55 93.2 42 97.7

Percentage of patients with CNI toxicity

Tremor was present in 39.3% (24) in 0-3 months, 34.4% (21) in 3-6 months, 25.4% (15) in 6-9 months and 20.9% (9) in more than 9 months. Hypertension was present in 39.3% (24) in 0-3 months, 31.1%

0 5 10 15 20 25 30 35 40

0-3 months 3-6 months 6-9 months >9 months

39.3

34.4

25.4

20.9

3.3 1.6 0 0

39.3

31.1

13.6

7

0 0 1.7

6.9

0 4.9 0 4.9 1.76.8 2.3

2.3

Tremor Paresthesia Hypertension Hypertrichosis Gumhypertrophy NODAT

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43

(19) in 3-6 months, 13.6% (6) in 6-9 months and 7% (3) in more than 9 months. Hypertrichosis and gum hypertrophy were present in few patients after 6 months of transplant. NODAT was present in 4.9% (93) in 0-3 months and 3-6 months, 6.8% (4) in 6-9 months and 2.3% (1) in more than 9 months.

During follow up improvement in graft dysfunction following tapering with calcineurin inhibitor was presumed to be due to calcineurin inhibitor toxicity after excluding rejection. Table 9 showed that graft dysfunction due to calcineurin inhibitor toxicity.

Table 9 Graft dysfunction

0-3 months 3-6 months 6-9 months >9 months

n % n % n % n %

Yes 23 37.7 14 23 22 37.3 3 7

No 38 62.3 47 77 37 62.7 40 93

Graft dysfunction was in 37.7% (23) in 0-3 months, 23% (14) in 3- 6 months, 37.3% (22) in 6-9 months, and 7% (3) in more than 9 months.

0 50 100

0-3 Months 3-6 Months 6-9 Months >9 Months

37.7 23 37.3

7

62.3 77

62.7

93

Yes No

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44

Whole blood trough(Co) level was done. Which shown in Table 10.

Table 10 Trough (Co) level

0-3 months 3-6 months 6-9 months >9 months

n % n % n % n %

Elevated CO

18 29.5 17 27.9 19 32.2 8 18.6 Normal Co 43 17.5 44 72.1 40 67.8 35 81.4

Elevated trough level was seen in 29.5% (18) in 0-3 months, 27.9%

(17) in 3-6 months, 32.2% (19) in 6-9 months and 18.6% (8) in more than 9 months.

Biopsy and histopathological examination was done only in small number of patients because of its invasiveness and procedure related

0 10 20 30 40 50 60 70 80 90

0-3 Months 3-6 Months 6-9 Months >9 Months

29.5 27.9 32.2

17.5 18.6

72.1 67.8

81.4

Elevated Co Normal Co

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45

complication, patient willingness and improvement with tapering of drugs. Biopsy features were shown in Table 11.

Table 11

Histopathological features of CNI toxicity 0-3

months

3-6 months

6-9

months >9 months No % No % No % No % Iso.vacuolization 6 9.8 5 8 2 3.4 0 0

Med. Hyalinosis 2 3.3 1 1.6 0 0 0 0

TMA 1 0 0 0 0 0 0 0

Glomerulosclerosis 0 0 0 0 0 0 0 0

Intertial fibrosis 0 0 1 0 0 0 0 0

Tubular atrophy 0 0 1 0 0 0 0 0

CNI Toxicity 6 9.8 5 8.2 2 3.4 0 0

In 0-3 months Six patients were found to have histopathological features of CNI toxicity. Isometric vacuolization was found in all biopsies, medullary hyalinosis and thrombotic microangiopathy were seen in two and one of those biopsies. In 3-6 months 5 patients shown histopathological evidence of CNI toxicity of which isometric vacuolization was present in all five biopsies and medullary hylinosis present in one biopsy. In 6-9 months, 2 patients were shown evidence of histopathological toxicity, isometric vacuolization was present in both of them.

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46

ANALYSIS

Statistical analyses were done by SPSS 20.6 software. Analysis was done in each post transplant age group. Factors analyzed were trough (Co) level (elevated trough (Co) level / normal trough (Co) level) versus clinical toxicity, tremor, paresthesia, hypertension, NODAT, graft dysfunction and histopathological toxicity. Hypertrichosis and gum hypertrophy were not analyzed because of its lower frequency in this study groups.

ANALYSIS AT 0-3 MONTHS

Clinical Toxicity vs Trough Level (0-3 months)

Elevated CO Normal CO P Clinical

toxicity

Present 14 23

0.077

Absent 4 20

0 5 10 15 20 25

Present Absent

14

4

23 20

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47

Analysis revealed that no significant correlation between the clinical toxicity and trough (Co) level.

Clinical features versus Trough (Co) level (0-3 months)

Elevated CO Normal CO P

Tremor Present 10 14 0.094

Absent 8 29

Paresthesia Present 1 1 0.518

Absent 17 42

Hypertension Present 6 18 0.534

Absent 12 25

NODAT Present 2 1 0.148

Absent 16 42

There is no significant correlation between tremor, paresthesia, hypertension, NODAT and trough (Co) level.

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48

Graft Dysfunction Vs Trough (Co) level (0-3 months)

Elevated CO Normal CO P Graft

dysfunction

Present 7 16

0.902

Absent 11 27

Analysis revealed that no significant correlation between the graft dysfunction and trough (Co) level.

0 2 4 6 8 10 12 14 16 18 20

Present Absent

7

11 16

20

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49

Histopathological Toxicity Vs Trough (Co) level (0-3 months) Elevated CO Normal CO P Histopathological

toxicity

Present 1 5

0.468

Absent 17 38

Analysis revealed that no significant correlation between the histopathological toxicity and trough (Co) level.

0 5 10 15 20 25 30 35 40

Present Absent

1

17

5

38

(61)

50

Clinical toxicity vs Trough (Co) level (3-6 months) Elevated CO Normal CO P Clinical

toxicity

Present 13 11

0.001

Absent 4 33

Analysis revealed that there was significant correlation between the clinical toxicity and trough (Co) level.

Clinical features Vs Trough (Co) level (3-6 months) Elevated CO Normal CO P

Tremor Present 13 8

0.001

Absent 4 36

Paresthesia Present 0 1

0.531

Absent 17 43

Hypertension Present 7 12

0.295

Absent 10 32

NODAT Present 1 2

0.829

Absent 16 42

Analysis revealed that there was significant correlation between tremor and elevated trough level. No significant correlation between paresthesia, hypertension, NODAT and elevated trough(Co) level.

0 10 20 30 40

Present Absent

13

11 4

33

(62)

51

Graft Dysfunction Vs Trough (Co) level (3-6 months) Elevated CO Normal CO P Graft

dysfunction

Present 4 10

0.947

Absent 13 34

Analysis revealed that no significant correlation between the graft dysfunction and trough (Co) level.

Histopathological Toxicity Vs Trough (Co) level (3-6 months) Elevated CO Normal CO P Histopathological

toxicity

Present 0 5

0.147

Absent 17 39

0 5 10 15 20 25 30 35

Present Absent

4

10 13

34

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52

Clinical Toxicity Vs Trough (Co) level (6-9 months) Elevated CO Normal CO P Clinical

toxicity

Present 11 4

0.001

Absent 8 36

Analysis revealed that there was significant correlation between the clinical toxicity and trough (Co) level.

0 20 40

Present Absent

0

17 5

39

0 20 40

Present Absent

11 4 8

36

(64)

53

Clinical Features Vs Trough (Co) level (6-9 months) Elevated CO Normal CO P

Tremor Present 12 3

0.001

Absent 7 37

Absent 0 0

0.005

Absent 13 38

NODAT Present 2 2

0.430

Absent 17 38

There was significant correlation between tremor and trough level.

No significant correlation between paresthesia, hypertension, NODAT and trough level.

Graft dysfunction Vs Trough (Co) level (6-9 months) Elevated CO Normal CO P Graft

dysfunction

Present 11 11

0.024

Absent 8 29

Analysis revealed that no significant correlation between the Graft dysfunction and trough (Co) level.

0 20 40

Present Absent

11 11 8

29

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54

Histopathological toxicity Vs Trough level (6-9 months) Elevated CO Normal CO P

HPE Present 2 0

0.37

Absent 17 40

0 5 10 15 20 25 30 35 40

Present Absent

2

17

0

40

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55

ANALYSIS AT >9 MONTHS

Clinical Toxicity Vs Trough (Co) level (>9 months)

Elevated CO Normal CO P Clinical

toxicity

Present 7 3

22.72

Absent 1 32

Analysis revealed that no significant correlation between the Clinical toxicity and trough (Co) level.

0 5 10 15 20 25 30 35

Present Absent

7

1 3

32

(67)

56

Clinical Features Vs Trough (Co) level (>9 months) Elevated CO Normal CO P

Tremor Present 6 3

17.36

Absent 2 32

Absent 0 0 0

4.91

Absent 6 34

NODAT Present 1 0

4.47

Absent 7 35

There was no significant correlation between tremor, paresthesia, hypertension, NODAT and trough level.

Graft Dysfunction Vs Trough (Co) level (>9 months)

Elevated CO Normal CO P Graft

dysfunction

Present 1 2

0.462

Absent 7 33

Analysis revealed that no significant correlation between the Graft dysfunction and trough (Co) level.

0 20 40

Present Absent

1 2 7

33

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57

The Correlation between clinical, trough (Co) level, graft function and histopathological toxicity

Clinical toxicity

Elevated CO

GDF Histopathological

Toxicity P 0-3

months 37 18 23 6 3.28

3-6months 24 17 14 5 3.81

6-9months 15 19 22 2 2.86

>9months 23 8 3 0 1.66

There was no significant correlation between clinical toxicity, elevated trough level, graft dysfunction, histopathological toxicity at 3-6, 6-9 and > 9 months.

0 5 10 15 20 25 30 35 40

0-3 months 3-6months 6-9months >9months

37

24

15

23

18 17

19

8 23

14

22

3

6 5

2

0

Clinical toxicity Elevated CO GDF Toxic histopathology

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58

TACROLIMUS VS CYCLOSPORINE

0-3MONTHS

Tacrolimus Cyclosporine P

Absent 22 15

Absent 29 30

Absent 17 20

Absent 27 31

Clinical toxicity Present 18 19

0.918

Absent 12 12

Graft

dysfunction

Present 9 14

0.222

Absent 21 17

Ttoxicity on HPE

Present 2 4

0.414

Absent 28 27

There was significant correlation between presence of tremor and Tacrolimus at 0-3 months. But there was no significant correlation between tacrolimus and cyclosporine in manifestation of paresthesia, hypertension, NODAT, overall clinical toxicity, graft dysfunction and histopathological toxicity.

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59

TACROLIMUS VS CYCLOSPORINE

3-6 MONTHS

Absent 20 20

Absent 29 31

Absent 20 22

Absent 29 29

0.918

Absent 18 19

Graft

dysfunction

Present 8 6

0.497

Absent 22 25

Toxicity on HPE

Present 1 4

0.173

Absent 29 27

There was no significant correlation between tacrolimus and cyclosporine in manifestation of tremor, paresthesia, hypertension, NODAT, overall clinical toxicity, graft dysfunction and histopathological toxicity.

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60

TACROLIMUS VS CYCLOSPORINE

6-9 MONTHS

Absent 23 21

Paresthesia Present 0 0 0

Absent 29 30

Absent 26 25

Absent 26 29

0.824

Absent 22 22

Graft

dysfunction

Present 7 15

0.404

Absent 22 15

Toxicity on HPE

Present 0 2

0.157

Absent 29 28

There was no significant correlation between tacrolimus and cyclosporine in manifestation of tremor, paresthesia, hypertension, NODAT, overall clinical toxicity, graft dysfunction and histopathological toxicity

A study on Clinical, Biochemical, Drug trough level and histopathological correlation of calcineurin inhibitor (CNI) toxicity in renal allograft recipient.